Benzyl phenyl ether derivative, preparation method therefor, and pharmaceutical composition and uses thereof

ABSTRACT

The present invention discloses a benzyl phenyl ether derivative, a preparation method therefor, and a pharmaceutical composition and uses thereof. Specifically, the invention relates to benzyl phenyl ether derivatives represented by formula (I), a pharmaceutically-acceptable salt thereof, a stereoisomer thereof, a preparation method therefor, a pharmaceutical composition containing the one or more compounds, and uses of the compounds in treating diseases related to PD-1/PD-L1 signal channels, such as cancers, infectious diseases and autoimmune diseases.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofinternational PCT application, PCT/CN2017/085417, filed May 23, 2017,which claims priority to Chinese Application No. 201610343960.7, filedMay 23, 2016.

FIELD OF THE INVENTION

The present invention discloses a benzyl phenyl ether derivative, apreparation method therefor, and a pharmaceutical composition and usesthereof. Specifically, the invention relates to benzyl phenyl etherderivatives represented by formula (I), a pharmaceutically-acceptablesalt thereof, a stereoisomer thereof, a preparation method therefor, apharmaceutical composition containing the one or more compounds, anduses of the compounds in treating diseases related to PD-1/PD-L1 signalchannels, such as cancers, infectious diseases and autoimmune diseases.

BACKGROUND OF THE INVENTION

With the deepening of research on cancer immunology, it has been foundthat the tumor microenvironment can protect tumor cells from beingrecognized and killed by the human immune system. The immune escape oftumor cells plays a very important role in tumor occurrence anddevelopment. In 2013, Science magazine ranked tumor immunotherapy as thefirst of the top ten breakthroughs, once again making immunotherapy a“focus” in the field of cancer treatment. Activation or inhibition ofimmune cells is regulated by positive and negative signals, whereinprogrammed cell death 1 (PD-1)/PD-1 ligand (PD-L1) is a negative immuneregulatory signal that inhibits the immune activity of tumor-specificCD8+ T cells and mediates immune escape.

Tumor cells evade the immune system by the binding of programmed celldeath ligand (PD-L1) produced on its surface to the PD-1 protein of Tcells. The tumor microenvironment induces high expression of PD-1molecules in infiltrating T cells, and tumor cells highly express PD-1ligands PD-L1 and PD-L2, resulting in continuous activation of the PD-1pathway in the tumor microenviroment. The inhibited T cells cannot findthe tumor so that it cannot signal the immune system to attack and killthe tumor cells. The PD-1 antibody against PD-1 or PD-L1 blocks thispathway by preventing the two proteins from binding and partiallyrestores the function of T cells, enabling them to kill tumor cells.

PD-1/PD-L1-based immunotherapy is a new generation high-profileimmunotherapy, aiming to use the body's own immune system to fighttumors. It has the potential to treat multiple types of tumors byblocking the PD-1/PD-L1 signaling pathway to induce apoptosis. Recently,a series of surprising studies have confirmed that PD-1/PD-L1 inhibitoryantibodies have strong anti-tumor activity against a variety of tumors,which is particularly eye-catching. On Sep. 4, 2014, Keytruda®(pembrolizumab) from Merck, USA, became the first FDA-approved PD-1monoclonal antibody for the treatment of advanced or unresectablemelanoma patients who were unresponsive for other medications.Currently, MSD is investigating the potential of Keytruda in more than30 different types of cancer, including various types of blood cancer,lung cancer, breast cancer, bladder cancer, stomach cancer, and head andneck cancer. On Dec. 22, 2014, pharmaceutical giant Bristol-Myers Squibbtook the lead in obtaining accelerated approval from the US Food andDrug Administration (FDA). Its anti-cancer immunotherapy drug nivolumabwas listed under the trade name Opdivo for the treatment of unresectableor metastatic melanoma patients who have not responded to other drugsand it is the second US-listed PD-1 inhibitor after MSD's Keytruda. OnMar. 4, 2015, FDA approved nivolumab for the treatment of metastaticsquamous non-small cell lung cancer that progressed duringplatinum-based chemotherapy or after chemotherapy. According to a PhaseIb KEYNOTE-028 study of the treatment of solid tumors by Keytruda(pembrolizumab) published by MSD, Keytruda treatment achieved a 28%overall response rate (ORR) in 25 patients with pleural mesothelioma(PM). And 48% of patients have stable disease and the disease controlrate has reached 76%. Patients with advanced Hodgkin's lymphoma (HL) whohad no treatment response to any of the approved drugs were able toachieve complete remission after receiving treatment with MSD's Keytrudaand Bristol-Myers' Opdvio. At the 2015 AACR Annual Meeting, Leisha A.Emens, MD, PhD, associate professor of oncology at the Johns HopkinsKimmel Cancer Center, reported that Roche's PD-L1 monoclonal antibodyMPDL3280A has a long-lasting effect in advanced triple-negative breastcancer.

Tumor immunotherapy is considered a revolution in cancer treatment aftertumor targeting therapy. However, the monoclonal antibody therapeuticdrug has its own defects: it is easily decomposed by proteases, so it isunstable within the body and cannot be taken orally; it is easy toproduce immune cross-reaction; the product quality is not easy tocontrol and the production technology is high; a large amount ofpreparation and purification is difficult, and the cost is high; it isinconvenient to use and it only can be injected or drip. Therefore,small molecule inhibitors of PD-1/PD-L1 interaction are a better choicefor tumor immunotherapy.

CONTENTS OF THE INVENTION

The technical problem to be solved by the present invention is toprovide a benzyl phenyl ether derivative with the structural formula (I)which inhibits the interaction of PD-1/PD-L1, and a stereoisomer thereofand a pharmaceutically acceptable salt thereof, and a preparation methodtherefor and medicament compositions thereof and their use in theprevention or treatment of a disease associated with the PD-1/PD-L1signaling pathway.

The technical solutions below are provided by the present invention inorder to solve the above technical problem.

The first aspect of the technical solution is to provide a benzyl phenylether derivative represented by formula (I), a stereoisomer thereof anda pharmaceutically-acceptable salt thereof:

wherein:

R₁ is selected from

R₂ is selected from hydrogen, cyano, methylsulfonyl, acetylamino,carbamoyl, dimethyl amino formyl (—CON(CH₃)₂), fluorine, chlorine,bromine, iodine, trifluoromethyl, C₁-C₅ alkyl and C₁-C₅ alkoxy;

R₃ is selected from substituted C₁-C₈ saturated alkylamino, substitutedC₂-C₆ unsaturated alkylamino, substituted N-containing C₂-C₆heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substitutedwith substituent(s) selected from hydrogen, fluorine, chlorine, bromine,iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino, C₁-C₆ alkylamino,acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino (—NH(C═NH)NH₂),ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂),sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino(—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxyl carbonyl, sulfydryl,imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, methoxy.

Preferable are benzyl phenyl ether derivatives, stereoisomers thereofand pharmaceutically acceptable salts thereof, wherein the compound isrepresented by formula (IA):

wherein:

R₁ is selected from

R₂ is selected from hydrogen, cyano, methylsulfonyl, acetylamino,carbamoyl, dimethyl amino formyl (—CON(CH₃)₂), fluorine, chlorine,bromine, iodine, trifluoromethyl, C₁-C₅ alkyl and C₁-C₅ alkoxy;

R₃ is selected from substituted C₁-C₈ saturated alkylamino, substitutedC₂-C₆ unsaturated alkylamino, substituted N-containing C₂-C₆heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substitutedwith substituent(s) selected from hydrogen, fluorine, chlorine, bromine,iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino, C₁-C₆ alkylamino,acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino (—NH(C═NH)NH₂),ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂),sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino(—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxyl carbonyl, sulfydryl,imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.

Preferable are benzyl phenyl ether derivatives, stereoisomers thereofand pharmaceutically acceptable salts thereof, wherein the compound isrepresented by formula (IA-1):

wherein:

R₂ is selected from hydrogen, cyano, methylsulfonyl, acetylamino,carbamoyl, dimethyl amino formyl (—CON(CH₃)₂), fluorine, chlorine,bromine, iodine, trifluoromethyl, C₁-C₅ alkyl and C₁-C₅ alkoxy;

R₃ is selected from substituted C₁-C₈ saturated alkylamino, substitutedC₂-C₆ unsaturated alkylamino, substituted N-containing C₂-C₆heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substitutedwith substituent(s) selected from hydrogen, fluorine, chlorine, bromine,iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino, C₁-C₆ alkylamino,acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino (—NH(C═NH)NH₂),ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂),sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino(—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxyl carbonyl, sulfydryl,imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.

Preferable are benzyl phenyl ether derivatives, stereoisomers thereofand pharmaceutically acceptable salts thereof, wherein the compound isrepresented by formula (IA-1a):

wherein:

R₃ is selected from substituted C₁-C₈ saturated alkylamino, substitutedC₂-C₆ unsaturated alkylamino, substituted N-containing C₂-C₆heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substitutedwith substituent(s) selected from hydrogen, fluorine, chlorine, bromine,iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino, C₁-C₆ alkylamino,acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino (—NH(C═NH)NH₂),ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂),sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino(—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxyl carbonyl, sulfydryl,imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.

Preferable are benzyl phenyl ether derivatives, stereoisomers thereofand pharmaceutically acceptable salts thereof, wherein the compound isrepresented by formula (IA-1b):

wherein:

R₃ is selected from substituted C₁-C₈ saturated alkylamino, substitutedC₂-C₆ unsaturated alkylamino, substituted N-containing C₂-C₆heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substitutedwith substituent(s) selected from hydrogen, fluorine, chlorine, bromine,iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino, C₁-C₆ alkylamino,acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino (—NH(C═NH)NH₂),ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂),sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino(—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxyl carbonyl, sulfydryl,imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.

Preferable are benzyl phenyl ether derivatives, stereoisomers thereofand pharmaceutically acceptable salts thereof, wherein the compound isrepresented by formula (IA-2):

wherein:

R₂ is selected from hydrogen, cyano, methylsulfonyl, acetylamino,carbamoyl, dimethyl amino formyl (—CON(CH₃)₂), fluorine, chlorine,bromine, iodine, trifluoromethyl, C₁-C₅ alkyl and C₁-C₅ alkoxy;

R₃ is selected from substituted C₁-C₈ saturated alkylamino, substitutedC₂-C₆ unsaturated alkylamino, substituted N-containing C₂-C₆heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substitutedwith substituent(s) selected from hydrogen, fluorine, chlorine, bromine,iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino, C₁-C₆ alkylamino,acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino (—NH(C═NH)NH₂),ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂),sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino(—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxyl carbonyl, sulfydryl,imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.

Preferable are benzyl phenyl ether derivatives, stereoisomers thereofand pharmaceutically acceptable salts thereof, wherein the compound isrepresented by formula (IA-2a):

wherein:

R₃ is selected from substituted C₁-C₈ saturated alkylamino, substitutedC₂-C₆ unsaturated alkylamino, substituted N-containing C₂-C₆heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substitutedwith substituent(s) selected from hydrogen, fluorine, chlorine, bromine,iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino, C₁-C₆ alkylamino,acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidine (—NH(C═NH)NH₂),ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂),sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino(—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxyl carbonyl, sulfydryl,imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.

Preferable are benzyl phenyl ether derivatives, stereoisomers thereofand pharmaceutically acceptable salts thereof, wherein the compound isrepresented by formula (IA-2b):

wherein:

R₃ is selected from substituted C₁-C₈ saturated alkylamino, substitutedC₂-C₆ unsaturated alkylamino, substituted N-containing C₂-C₆heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substitutedwith substituent(s) selected from hydrogen, fluorine, chlorine, bromine,iodine, hydroxy, C₁-C₆ alkyl, C₁-C₆ alkoxy, amino, C₁-C₆ alkylamino,acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidine (—NH(C═NH)NH₂),ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂),sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino(—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxyl carbonyl, sulfydryl,imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.

Preferable are benzyl phenyl ether derivatives, stereoisomers thereofand pharmaceutically acceptable salts thereof, wherein the compound isrepresented in the above formulae, wherein R₃ is selected from:

wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl,pentyl, hexyl, heptyl, octyl; and

X is selected from hydrogen, fluorine, chlorine, bromine, methyl,ethenyl, and trifluoromethyl.

The benzyl phenyl ether derivative of the above formulae, a stereoisomerthereof and a pharmaceutically acceptable salt thereof, arecharacterized in that, the pharmaceutically acceptable salt comprises asalt formed with an inorganic acid, a salt formed with an organic acidsalt, alkali metal ion salt, alkaline earth metal ion salt or a saltformed with organic base which provides a physiologically acceptablecation, and an ammonium salt.

Further, said inorganic acid is selected from hydrochloric acid,hydrobromic acid, phosphoric acid or sulfuric acid; said organic acid isselected from methanesulfonic acid, p-toluenesulfonic acid,trifluoroacetic acid, citric acid, maleic acid, tartaric acid, fumaricacid, citric acid or lactic acid; said alkali metal ion is selected fromlithium ion, sodium ion, potassium ion; said alkaline earth metal ion isselected from calcium ion, magnesium ion; said organic base, whichprovides physiologically acceptable cation, is selected frommethylamine, dimethylamine, trimethylamine, piperidine, morpholine ortris(2-hydroxyethyl)amine.

The second aspect of the present invention provides a method forpreparing the compounds of the first aspect.

For the preparation of the compounds of the formula (I), according toits structure, the preparation method is divided into five steps.

-   -   (a) 2-bromo-3-iodotoluene 1 and benzene boronic acid or        substituted benzene boronic acid or boric acid ester of benzene        or substituted benzene as starting materials are reacted by        suzuki coupling reaction to obtain Intermediate compound 2;    -   (b) intermediate 2 as a starting material is subjected to        bromination of the methyl group by a bromination reagent to give        the bromo intermediate 3;    -   (c) intermediate 3 as a starting material is reacted with        2,4-dihydroxy-X-substituted benzaldehyde under basic conditions        to obtain benzyl aryl ether intermediate 4;    -   (d) intermediate 4 as a starting material is reacted with a        benzyl halide, or substituted benzyl halide under basic        conditions to give intermediate compound 5;    -   (e) an aldehyde group-containing intermediate compound 5 as a        starting material is condensed with an amino group- or an imino        group-containing HR₃ and the resultant product is reduced to        obtain the target compound I.

R₁, R₂, R₃ and X each is defined as described in the first aspect.

In addition, the starting materials and intermediates in the abovereaction are obtained easily, and each step reaction can be performedeasily according to the reported literature or by a skilled worker inthe art by a conventional method in organic synthesis. The compound offormula I may exist in solvated or unsolvated forms, and crystallizationfrom different solvents may result in different solvates. Thepharmaceutically acceptable salts of the formula (I) include differentacid addition salts, such as the acid addition salts of the followinginorganic or organic acids: hydrochloric acid, hydrobromic acid,phosphoric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonicacid, trifluoroacetic acid, citric acid, maleic acid, tartaric acid,fumaric acid, citric acid, lactic acid. The pharmaceutically acceptablesalts of formula I also include various alkali metal salts such aslithium, sodium, potassium salts; various alkaline-earth metal saltssuch as calcium, magnesium salts and ammonium salts; and various organicbase salts which provide physiologically acceptable cations, such asmethylamine, dimethylamine, trimethylamine, piperidine, morpholine saltsand tris(2-hydroxyethyl)amine salts. All of these salts within the scopeof the invention can be prepared by conventional methods. During thepreparation of the compounds of the formula (I) and their solvates orsalts, polycrystalline or eutectic may occur under differentcrystallization conditions.

The third aspect of the present invention provides a pharmaceuticalcomposition comprising which includes the benzyl phenyl ether derivativeof the first aspect of the present invention and a stereoisomer thereof,and the pharmaceutically acceptable salt as an active ingredient and apharmaceutically acceptable carrier or excipient.

The invention further relates to a pharmaceutical composition comprisinga compound of the invention as an active ingredient. The pharmaceuticalcomposition can be prepared according to methods well known in the art.Any dosage form suitable for human or animal use can be prepared bycombining a compound of the invention with one or more pharmaceuticallyacceptable excipients and/or adjuvants in solid or liquid. The contentof the compound of the present invention in its pharmaceuticalcomposition is usually from 0.1 to 95% by weight.

The compound of the present invention or the pharmaceutical compositioncontaining the same can be administered in a unit dosage form, viaenteral or parenteral route, such as oral, intravenous, intramuscular,subcutaneous, nasal, oral mucosa, eye, lung and the respiratory tract,skin, vagina, rectum, etc.

The dosage form can be a liquid dosage form, a solid dosage form or asemi-solid dosage form. Liquid dosage forms can be solution (includingtrue solution and colloidal solution), emulsion (including o/w type, w/otype and double emulsion), suspension, injection (including waterinjection, powder injection and infusion), eye drops, nasal drops,lotions, liniments, etc.; solid dosage forms may be tablets (includingordinary tablets, enteric tablets, lozenges, dispersible tablets,chewable tablets, effervescent tablets, orally disintegrating tablets),capsules (including hard capsules, soft capsules, enteric capsules),granules, powders, pellets, dropping pills, suppositories, films,patches, gas (powder) sprays, sprays, etc.; semi-solid dosage forms canbe ointments, gel, paste, etc.

The compounds of the present invention can be formulated into commonpreparations, as well as sustained release preparations, controlledrelease preparations, targeted preparations, and various microparticledelivery systems.

In order to form tablets of the compound of the present invention into,various excipients known in the art, including diluents, binders,wetting agents, disintegrating agents, lubricants, and glidants, can beused widely. The diluent may be starch, dextrin, sucrose, glucose,lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose,calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.;the wetting agent may be water, ethanol, or isopropanol, etc.; thebinder may be starch syrup, dextrin, syrup, honey, glucose solution,microcrystalline cellulose, acacia mucilage, gelatine, sodiumcarboxymethyl cellulose, methyl cellulose, hydroxypropylmethylcellulose, ethyl cellulose, acrylic resin, carbomer,polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can bedry starch, microcrystalline cellulose, low-substituted hydroxypropylcellulose, cross-linked poly vinyl pyrrolidone, croscarmellose sodium,sodium carboxymethyl starch, sodium hydrogencarbonate and citric acid,polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfonate,etc.; lubricant and glidant may be talc, silica, stearate, tartaricacid, liquid paraffin, polyethylene glycol, etc.

Tablets may also be further formulated into coated tablets such as sugarcoated tablets, film-coated tablets, enteric coated tablets, or bilayertablets and multilayer tablets.

In order to prepare the dose unit as a capsule, the active ingredientcompound of the present invention may be mixed with a diluent, aglidant, and the mixture may be directly placed in a hard capsule or asoft capsule. The active ingredient can also be formulated into agranule or pellet with a diluent, a binder, a disintegrant, and thenplaced in a hard or soft capsule. Various diluents, binders, wettingagents, disintegrating agents and glidants for preparing the tablets ofthe compound of the invention can also be used to prepare the capsulesof the compound of the invention.

In order to prepare the compound of the present invention as aninjection, water, ethanol, isopropanol, propylene glycol or theirmixture may be used as a solvent. In addition, an appropriate amount ofa solubilizing agent, a co-solvent, a pH adjusting agent, and an osmoticpressure adjusting agent which are commonly used in the art can beadded. The solubilizing agent or co-solvent may be poloxamer, lecithin,hydroxypropyl-β-cyclodextrin, etc.; the pH adjusting agent may bephosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; osmoticpressure regulating agent may be sodium chloride, mannitol, glucose,phosphate, acetate, etc. For preparing a lyophilized powder injection,mannitol, glucose and so on may also be added as a proppant.

In addition, coloring agents, preservatives, perfumes, flavoring agentsor other additives may also be added to the pharmaceutical preparationsas needed.

The compound or pharmaceutical composition of the present invention canbe administered by any known administration method for the purpose ofadministration and enhancing the therapeutic effect.

The dosage of the compound or the pharmaceutical composition of thepresent invention can be administered in a wide range depending on thenature and severity of the disease to be prevented or treated, theindividual condition of the patient or animal, the route ofadministration and the dosage form, etc. In general, a suitable dailydose of the compound of the invention will range from 0.001 to 150 mg/kgbody weight, preferably from 0.01 to 100 mg/kg body weight. The abovedosages may be administered in a single dosage unit or in divided doseunits depending on the clinical experience of the physician and thedosage regimen including the use of other therapeutic means.

The compounds or compositions of the invention may be administered aloneor in combination with other therapeutic or symptomatic agents. When thecompound of the present invention synergizes with other therapeuticagents, its dosage should be adjusted according to the actual situation.

The fourth aspect of the present invention provides a benzyl phenylether derivative, or a stereoisomer thereof, or a pharmaceuticallyacceptable salt thereof, which are used for the preparation of amedicament useful for preventing and/or treating a disease associatedwith the PD-1/PD-L1 signaling pathway.

The disease associated with the PD-1/PD-L1 signaling pathway is selectedfrom cancer, infectious diseases, and autoimmune diseases. The cancer isselected from skin cancer, lung cancer, urinary tumor, hematologicaltumor, breast cancer, glioma, digestive system tumor, reproductivesystem tumor, lymphoma, nervous system tumor, brain tumor, head and neckcancer. The infectious disease is selected from bacterial infection andviral infection. The autoimmune disease is selected from organ-specificautoimmune disease, systemic autoimmune disease, wherein theorgan-specific autoimmune disease includes chronic lymphocyticthyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus,myasthenia gravis, ulcerative colitis, malignant anemia with chronicatrophic gastritis, pulmonary hemorrhagic nephritis syndrome, primarybiliary cirrhosis, multiple cerebrospinal sclerosis, and acuteidiopathic polyneuritis. And the systemic autoimmune diseases includerheumatoid arthritis, systemic lupus erythematosus, systemic vasculitis,scleroderma, pemphigus, dermatomyositis, mixed connective tissuedisease, autoimmune hemolytic anemia.

Beneficial Technical Effects:

The compounds of the present invention have high inhibitory activity onPD-1/PD-L1 interaction, much higher than the reported compounds. Theyhave strong ability of binding PD-L1 protein, even stronger than thereported antibodies of PD-L1. These compounds also have the ability torelieve the inhibition of IFN-γ by PD-L1. The pharmacodynamic studies invivo show that the compounds can significantly inhibit the growth ofsubcutaneous tumors in both tumor volume and weight. The number oflymphocytes in blood and spleen of mice can be increased obviously.

EXAMPLES

The invention is further illustrated by the following examples; however,the invention is not limited by the illustrative examples set hereinbelow.

Measuring instrument: Nuclear magnetic resonance spectroscopy wascarried out by using a Vaariaan Mercury 300 nuclear magnetic resonanceapparatus. Mass spectrometry was performed by using ZAD-2F massspectrometer and VG300 mass spectrometer.

Example 1N-acetylaminoethyl-4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzylamine

(1) 2-bromo-3-methyl-1,1′-biphenyl

To a 50 ml flask was added 2-bromo-3-iodotoluene (350 mg) anddioxane/water with stirring. The solution was bubbled with argon for 10min to remove dissolved oxygen. Then, phenylboronic acid (172.65 mg),cesium carbonate (961.2 mg), and triphenylphosphine palladium (40.91 mg)were sequentially added. The mixture was stirred for 12 h at 80-100° C.under argon protection. The reaction was stopped. After cooling to roomtemperature, the mixture was filtered with diatomaceous earth. Thefiltrate was concentrated under reduced pressure and extracted withwater and ethyl acetate for three times. The organic phase was combined,washed with saturated brine, and dried over anhydrous sodium sulfate.The organic layer was filtered and concentrated in vacuo. The residuewas subjected to silica gel column chromatography (petroleum ether), toafford a colorless oil (221 mg). ¹H NMR (400 MHz, DMSO-d₆), δ 7.49-7.29(m, 7H, Ar—H), 7.14 (d, 1H, Ar—H), 2.42 (s, 3H, Ar—CH₃).

(2) 2-Bromo-3-(bromomethyl)-1,1′-biphenyl

2-Bromo-3-methyl-1,1′-biphenyl (234 mg) as a starting material was takenand dissolved in 20 ml CCl₄ in a 100 ml flask. To this solution wasadded NBS (178 mg) while stirring. And the mixture was warmed to 80° C.and refluxed. Then benzoyl peroxide (4 mg) was added, and after 2 h,benzoyl peroxide (4 mg) was added again, and the mixture was stirred foranother 2 h. The reaction was stopped. After cooling to roomtemperature, the mixture was quenched with water, extracted withdichloromethane and water. The organic phase was washed with saturatedbrine, and dried over anhydrous sodium sulfate. The organic layer wasfiltered and concentrated in vacuo to afford a yellow oil (192 mg),which was used for the next step without further purification.

(3) 4-(2-bromo-3-phenylbenzyloxy)-2-hydroxybenzaldehyde

2,4-dihydroxybenzaldehyde (73.94 mg) was taken and dissolved in 6 mlanhydrous acetonitrile in a 50 ml flask, and then sodium hydrogencarbonate (98.88 mg) was added. After stirring at room temperature for40 min, 2-bromo-3-(bromomethyl)-1,1′-biphenyl (192 mg, dissolved in 8 mlDMF) was slowly added to the reaction mixture via a constant pressuredropping funnel, and heated to reflux until the reaction was completed.After cooling to room temperature, the mixture was extracted with waterand ethyl acetate. The organic phase was washed with saturated brine,and dried over anhydrous sodium sulfate, then filtrated and concentratedin vacuo. The crude residue was purified by silica gel columnchromatography to afford a white solid (152 mg). ¹H NMR (400 MHz,DMSO-d₆) δ 10.99 (s, 1H, —OH), 10.03 (s, 1H, —CHO), 7.64 (d, 1H, Ar—H),7.57 (d, 1H, Ar—H), 7.45 (m, 4H, Ar—H), 7.37 (d, 3H, Ar—H), 6.67 (d, 1H,Ar—H), 6.59 (s, 1H, Ar—H), 5.25 (s, 2H, —CH₂—).

(4) 4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy) benzaldehyde

4-(2-bromo-3-phenylbenzyloxy)-2-hydroxybenzaldehyde (100 mg) wasdissolved in 6 ml DMF in a 50 ml flask, and then cesium carbonate(127.53 mg) was added. After stirring at room temperature for 15 min, asolution of 3-cyanobenzyl bromide (76.65 mg) in DMF (4 ml) was addeddropwise. After the mixture was stirred at 80° C. for 2 h, the reactionwas stopped. After cooling to room temperature, the mixture wasextracted with water and ethyl acetate. The organic phase was washedwith saturated brine, and dried over anhydrous sodium sulfate, thenfiltrated and concentrated in vacuo. The crude residue was purified bysilica gel column chromatography to afford a white solid (70 mg). ¹H NMR(400 MHz, DMSO-d₆) δ 10.26 (s, 1H, —CHO), 8.00 (s, 1H, Ar—H), 7.83 (dd,2H, Ar—H), 7.72 (d, 1H, Ar—H), 7.61 (t, 2H, Ar—H), 7.55-7.23 (m, 7H,Ar—H), 6.95 (s, 1H, Ar—H), 6.81 (d, 1H, Ar—H), 5.35 (s, 2H, —CH₂—), 5.30(s, 2H, —CH₂—).

(5)N-acetylaminoethyl-4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzylamine

4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy) benzaldehyde (50.8mg) was dissolved in 5 ml DMF, and then 2-acetamidoethylamine (31.25 mg)and acetic acid glacial (36.75 mg) were added. After stirring at roomtemperature for 20 min, sodium cyanoborohydride (19.23 mg) was added andthe mixture was stirred at 25° C. for 14 h. The reaction was stopped.The mixture was extracted with water and ethyl acetate. The organicphase was washed with saturated brine, and dried over anhydrous sodiumsulfate, then filtrated and concentrated in vacuo. The residue waspurified by silica gel column chromatography to afford a white solid (35mg). ¹H NMR (400 MHz, DMSO-d₆) δ 8.01 (s, 1H, —CONH—), 7.96 (s, 1H,Ar—H), 7.82 (dd, 2H, Ar—H), 7.59 (dd, 2H, Ar—H), 7.43 (dd, 4H, Ar—H),7.35 (t, 4H, Ar—H), 6.81 (s, 1H, Ar—H), 6.68 (d, 1H, Ar—H), 5.23 (s, 2H,—CH₂—), 5.18 (s, 2H, —CH₂—), 3.96 (s, 2H, —CH₂—), 3.28-3.21 (m, 2H,—CH₂—), 2.80 (t, 2H, —CH₂—), 1.89 (s, 1H, —NH—), 1.78 (s, 3H, —CH₃). MS(FAB): 585 (M+1).

Example 2: N-(4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy) benzyl)serine

The procedure was the same as in Example 1, except that L-serine wasused in place of 2-acetamidoethylamine to afford a white solid. ¹H NMR(400 MHz, DMSO-d₆) δ 8.00 (s, 1H, Ar—H), 7.89 (d, 1H, Ar—H), 7.79 (d,1H, Ar—H), 7.58 (dd, 2H, Ar—H), 7.53-7.29 (m, 8H, Ar—H), 6.81 (s, 1H,Ar—H), 6.67 (d, 1H, Ar—H), 5.23 (s, 2H, —CH₂—), 5.18 (s, 2H, —CH₂—),4.14-3.97 (m, 2H, —CH₂—), 3.74 (dd, 1H, —CH₂—), 3.62 (dd, 1H, —CH₂—),3.17 (t, 1H, —CH—). MS (FAB): 588 (M+1). [α]^(D) ₂₀=−16 (C=0.18,CH₂Cl₂).

Example 3N-Ethyl-N-hydroxylethyl-4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzylamine

The procedure was the same as in Example 1, except that2-(ethylamino)ethanol was used in place of 2-acetamidoethylamine toafford a pale yellow solid powder. ¹H NMR (400 MHz, DMSO-d₆) δ 9.33 (s,1H, —OH), 8.01 (s, 1H, —ArH), 7.84 (dd, 2H, —ArH), 7.61 (dd, 2H, —ArH),7.55-7.28 (m, 8H, —ArH), 6.88 (s, 1H, —ArH), 6.74 (d, 1H, —ArH), 5.24(s, 2H, —CH₂—), 5.21 (s, 2H, —CH₂—), 4.25 (s, 2H, —CH₂—), 3.69 (s, 2H,—CH₂—), 3.06 (s, 4H, —CH₂—), 1.18 (m, 3H, —CH₃). MS (FAB): 572 (M+1).

Example 4: N-(4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzyl)proline

The procedure was the same as in Example 1, except that proline was usedin place of 2-acetamidoethylamine to afford a pale yellow solid powder.¹H NMR (400 MHz, DMSO-d₆) δ 8.00 (s, 1H, —ArH), 7.89 (d, 1H, —ArH), 7.80(d, 1H, —ArH), 7.60 (dd, 2H, —ArH), 7.40 (m, 8H, —ArH), 6.82 (s, 1H,—ArH), 6.68 (d, 1H, —ArH), 5.31-5.22 (m, 2H, —CH₂—), 5.19 (s, 2H,—CH₂—), 4.19-4.01 (m, 2H, —CH₂—), 3.53 (m, 1H, —CH—), 3.23 (m, 1H,—CH₂—), 2.83 (m, 1H, —CH₂—), 2.09 (t, 1H, —CH₂—), 1.95 (t, 1H, —CH₂—),1.83 (s, 1H, —CH₂—), 1.66 (m, —CH₂—). MS (FAB): 598 (M+1).

Example 5N-hydroxylethyl-4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzylamine

The procedure was the same as in Example 1, except that 2-aminoethanolwas used in place of 2-acetamidoethylamine to afford a pale yellow solidpowder. ¹H NMR (400 MHz, DMSO-d₆) δ 8.91 (s, 1H, —OH), 7.99 (s, 1H,—ArH), 7.84 (dd, 2H, —ArH), 7.60 (dd, 2H, —ArH), 7.54-7.28 (m, 8H,—ArH), 6.84 (d, 1H, —ArH), 6.71 (dd, 1H, —ArH), 5.24 (s, 2H, —CH₂—),5.20 (s, 2H, —CH₂—), 4.09 (s, 2H, —CH₂—), 3.65 (d, 2H, —CH₂—), 2.91 (t,2H, —CH₂—). MS (FAB): 544 (M+1).

Example 6: N-(4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzyl)alanine

The procedure was the same as in Example 1, except that alanine was usedin place of 2-acetamidoethylamine to afford a pale yellow solid powder.¹H NMR (400 MHz, DMSO-d₆) δ 7.99 (s, 1H, —ArH), 7.84 (dd, 2H, —ArH),7.59 (dd, 2H, —ArH), 7.40 (dq, 8H, —ArH), 6.90-6.56 (m, 2H, —ArH), 5.22(s, 2H, —CH₂—), 5.18 (s, 2H, —CH₂—), 3.99 (d, 2H, —CH₂—), 1.86 (s, 1H,—CH—), 1.26 (s, 3H, —CH₃). MS (FAB): 572 (M+1).

Example 7: N-(4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzyl)methionine

The procedure was the same as in Example 1, except that methionine wasused in place of 2-acetamidoethylamine to afford a pale yellow solidpowder. ¹H NMR (400 MHz, DMSO-d₆) δ 7.99 (s, 1H, —ArH), 7.88 (d, 1H,—ArH), 7.80 (d, 1H, —ArH), 7.60 (dd, 2H, —ArH), 7.47 (d, 3H, —ArH), 7.43(d, 1H, —ArH), 7.36 (m, 4H, —ArH), 6.80 (s, 1H, —ArH), 6.67 (d, 1H,—ArH), 5.23 (s, 2H, —CH₂—), 5.19 (s, 2H, —CH₂—), 3.90 (q, 2H, —CH₂—),3.21 (d, 1H, —CH—), 2.56 (d, 1H, —CH₂—), 2.03 (s, 1H, —CH₂—), 1.98 (s,3H, —CH₃), 1.94-1.87 (m, 1H, —CH₂—), 1.87-1.80 (m, 1H, —CH₂—). MS (FAB):632 (M+1).

Example 8: N-(4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy) benzyl)threonine

The procedure was the same as in Example 1, except that threonine wasused in place of 2-acetamidoethylamine to afford a white solid powder.¹H NMR (400 MHz, DMSO-d₆) δ 8.01 (s, 1H, —ArH), 7.90-7.86 (t, 1H, —ArH),7.84-7.81 (t, 1H, —ArH), 7.75-7.73 (d, 1H, —ArH), 7.63-7.58 (m, 2H,—ArH), 7.50-7.46 (m, 3H, —ArH), 7.43-7.42 (d, 1H, —ArH), 7.39-7.35 (m,3H, —ArH), 5.36-5.32 (d, 2H, —CH₂—), 5.24-5.23 (m, 2H, —CH₂—), 3.98-3.96(m, 1H, —CH—), 3.88-3.86 (m, 1H, —CH—), 1.13 (d, 3H, —CH₃). MS (FAB):602 (M+1).

Example 9N-(tetrahydro-2H-pyran-4-yl)-4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzylamine

The procedure was the same as in Example 1, except thattetrahydro-2H-pyran-4-amine was used in place of 2-acetamidoethylamineto afford a pale yellow solid powder. ¹H NMR (400 MHz, DMSO-d₆) δ 8.12(d, 1H, —ArH), 7.95 (m, 3H, —ArH), 7.73 (m, 2H, —ArH), 7.65-7.41 (m, 7H,—ArH), 6.96 (d, 1H, —ArH), 6.83 (d, 1H, —ArH), 5.33 (s, 4H, —CH₂—), 4.13(d, 2H, —CH₂—), 3.94 (d, 2H, —CH₂—), 3.37 (d, 2H, —CH₂—), 3.23 (s, 1H,—CH—), 2.02 (d, 2H, —CH₂—), 1.61 (d, 2H, —CH₂—). MS (FAB): 584 (M+1).

Example 10: N-[4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzyl] morpholine Hydrochloride

The procedure was the same as in Example 1, except that morpholine wasused in place of 2-acetamidoethylamine to afford a pale yellow solidpowder. ¹H NMR (400 MHz, DMSO-d₆) δ 11.20 (s, 1H, H—Cl), 8.03 (d, 1H,—ArH), 7.94-7.86 (m, 1H, —ArH), 7.81 (t, 2H, —ArH), 7.61 (m, 3H, —ArH),7.54-7.44 (m, 2H, —ArH), 7.42 (d, 1H, —ArH), 7.38 (d, 2H, —ArH), 7.23(s, 1H, —ArH), 6.89 (d, 1H, —ArH), 6.75 (t, 1H, —ArH), 5.27 (d, 2H,—CH₂—), 5.23 (d, 2H, —CH₂—), 4.25 (t, 2H, —CH₂—), 3.58 (t, 2H, —CH₂—),3.04 (s, 2H, —CH₂—), 2.98-2.82 (m, 2H, —CH₂—), 2.59 (m, 2H, —CH₂—). MS(FAB): 606 (M+1).

Example 11N-hydroxylethyl-4-(2-bromo-3-(3,4-dimethoxyphenyl)benzyloxy)-2-(3-cyanobenzyloxy) benzylamine

(1) 2-Bromo-3-(3,4-dimethoxyphenyl)toluene

The procedure was the same as in Example 1, except that2-(3,4-dimethoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane was usedin place of phenylboronic acid, [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) was used in place of triphenylphosphinepalladium, potassium carbonate was used in place of cesium carbonate toafford 2-bromo-3-(3,4-dimethoxyphenyl)toluene. ¹H NMR (400 MHz,Chloroform-d) δ 7.22 (d, 2H, —ArH), 7.15 (q, 1H, —ArH), 6.93 (s, 3H,—ArH), 3.91 (d, 6H, —OCH₃), 2.49 (s, 3H, —CH₃).

(2) 4-(2-bromo-3-(3,4-dimethoxyphenyl)benzyloxy)-2-hydroxybenzaldehyde

The procedure was the same as in Example 1, except that2-bromo-3-(3,4-dimethoxyphenyl)toluene was used in place of2-bromo-3-methyl-1,1′-biphenyl to effect bromination; and the bromide,without further purification, was reacted directly with 2,4-dihydroxybenzaldehyde to afford a white solid. ¹H NMR (400 MHz,DMSO-d₆) δ 10.99 (s, 1H, —OH), 10.03 (s, 1H, —CHO), 7.64 (d, 1H, —ArH),7.53 (d, 1H, —ArH), 7.46 (t, 1H, —ArH), 7.37 (d, 1H, —ArH), 7.02 (d, 1H,—ArH), 6.94 (s, 1H, —ArH), 6.92-6.85 (m, 1H, —ArH), 6.67 (d, 1H, —ArH),6.59 (s, 1H, —ArH), 5.24 (s, 2H, —CH₂—), 3.77 (s, 6H, —OCH₃).

(3) 4-(2-bromo-3-(3,4-dimethoxyphenyl)benzyloxy)-2-(3-cyanobenzyloxy)benzaldehyde

The procedure was the same as in Example 1, except that4-(2-bromo-3-(3,4-dimethoxyphenyl)benzyloxy)-2-hydroxybenzaldehyde wasused in place of 4-(2-bromo-3-phenylbenzyloxy)-2-hydroxybenz aldehyde toafford a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.28 (s, 1H, —CHO),8.02 (s, 1H, —ArH), 7.87 (d, 1H, —ArH), 7.83 (d, 1H, —ArH), 7.74 (d, 1H,—ArH), 7.64 (t, 1H, —ArH), 7.58 (d, 1H, —ArH), 7.48 (t, 1H, —ArH), 7.40(d, 1H, —ArH), 7.04 (d, 1H, —ArH), 6.96 (s, 2H, —ArH), 6.91 (d, 1H,—ArH), 6.82 (d, 1H, —ArH), 5.37 (s, 2H, —CH₂—), 5.32 (s, 2H, —CH₂—),3.81 (s, 3H, —OCH₃), 3.78 (s, 3H, —OCH₃).

(4)N-hydroxylethyl-4-(2-bromo-3-(3,4-dimethoxyphenyl)benzyloxy)-2-(3-cyanobenzyloxy)benzylamine

The procedure was the same as in Example 1, except that4-(2-bromo-3-(3,4-dimethoxyphenyl)benzyloxy)-2-(3-cyanobenzyloxy)benzaldehyde was used in place of4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzaldehyde,2-aminoethanol was used in place of 2-acetamidoethylamine to afford awhite solid powder. ¹H NMR (400 MHz, DMSO-d₆) δ 8.52 (s, 1H, —OH), 7.98(s, 1H, —ArH), 7.83 (dd, 2H, —ArH), 7.61 (t, 1H, —ArH), 7.54 (d, 1H,—ArH), 7.45 (m, 1H, —ArH), 7.37 (dd, 2H, —ArH), 7.02 (d, 1H, —ArH), 6.93(d, 1H, —ArH), 6.91-6.81 (m, 2H, —ArH), 6.72 (dd, 1H, —ArH), 5.24 (s,2H, —CH₂—), 5.20 (s, 2H, —CH₂—), 4.10 (s, 2H, —CH₂—), 3.79 (s, 3H,—OCH₃), 3.76 (s, 3H, —OCH₃), 3.63 (q, 2H, —CH₂—), 2.92 (t, 2H, —CH₂—).MS (FAB): 604 (M+1).

Example 12: MethylN-(4-(2-bromo-3-(3,4-dimethoxyphenyl)benzyloxy)-2-(3-cyanobenzyloxy)benzyl) serinate

The procedure was the same as in Example 11, except that methyl serinatewas used in place of 2-aminoethanol, to afford a pale yellow solidpowder. ¹H NMR (400 MHz, DMSO-d₆) δ 7.93 (s, 1H, —ArH), 7.79 (s, 2H,—ArH), 7.60 (t, 1H, —ArH), 7.53 (d, 1H, —ArH), 7.44 (t, 1H, —ArH), 7.34(d, 1H, —ArH), 7.22 (d, 1H, —ArH), 7.02 (d, 1H, —ArH), 6.94 (s, 1H,—ArH), 6.89 (d, 1H, —ArH), 6.75 (s, 1H, —ArH), 6.61 (d, 1H, —ArH), 5.19(s, 2H, —CH₂—), 5.15 (s, 2H, —CH₂—), 4.88 (s, 1H, —NH—), 3.79 (s, 3H,—OCH₃), 3.76 (s, 3H, —OCH₃), 3.65 (d, 1H, —CH₂—), 3.57 (d, 2H, —CH₂—),3.55 (s, 3H, —OCH₃), 3.35 (m, 1H, —CH₂—). MS (FAB): 662 (M+1).

Example 13N-(4-(2-bromo-3-(3,4-dimethoxyphenyl)benzyloxy)-2-(3-cyanobenzyloxy)benzyl)serine

MethylN-(4-(2-bromo-3-(3,4-dimethoxyphenyl)benzyloxy)-2-(3-cyanobenzyloxy)benzyl) serinate (50 mg) was dissolved in methanol/H₂O (10 ml/1 ml), andthen lithium hydroxide monohydrate (90 mg) was added. After beingrefluxed for 2 h, the reaction was cooled to the room temperature. Thereaction was stopped. And a few drops of acetic acid were added to themixture in an ice bath to adjust the pH to 5-6. The mixture wasextracted by water and ethyl acetate. The organic phase was combined,washed with saturated brine, and dried over anhydrous sodium sulfate.The organic layer was then filtered and concentrated in vacuo. Theresidue was washed by diethyl ether to afford an off-white solid powder(25 mg). ¹H NMR (400 MHz, DMSO-d₆) δ 8.00 (s, 1H, —ArH), 7.90 (d, 1H,—ArH), 7.81 (d, 1H, —ArH), 7.61 (t, 1H, —ArH), 7.55 (d, 1H, —ArH), 7.45(t, 1H, —ArH), 7.37 (d, 2H, —ArH), 7.04 (d, 1H, —ArH), 6.95 (s, 1H,—ArH), 6.90 (d, 1H, —ArH), 6.82 (s, 1H, —ArH), 6.74-6.61 (m, 1H, —ArH),5.25 (s, 2H, —CH₂—), 5.19 (s, 2H, —CH₂—), 4.09 (s, 2H, —CH₂—), 3.85 (s,1H, —CH—), 3.81 (s, 3H, —OCH₃), 3.73 (s, 3H, —OCH₃), 3.72-3.62 (m, 1H,—CH₂—), 3.38 (q, 1H, —CH₂—). MS (FAB): 648 (M+1).

Example 14N-acetylaminoethyl-4-(2-bromo-3-(3,4-dimethoxyphenyl)benzyloxy)-2-(3-cyanobenzyloxy) benzylamine

The procedure was the same as in Example 11, except that2-acetamidoethylamine was used in place of 2-aminoethanol to afford anoff-white solid powder. ¹H NMR (400 MHz, DMSO-d₆) δ 8.67 (s, 1H, —NH—),8.11 (t, 1H, —NHCO—), 7.99 (s, 1H, —ArH), 7.83 (dd, 2H, —ArH), 7.61 (t,1H, —ArH), 7.53 (d, 1H, —ArH), 7.44 (t, 1H, —ArH), 7.37 (dd, 2H, —ArH),7.02 (d, 1H, —ArH), 6.93 (s, 1H, —ArH), 6.89 (d, 1H, —ArH), 6.84 (s, 1H,—ArH), 6.72 (d, 1H, —ArH), 5.25 (s, 2H, —CH₂—), 5.19 (s, 2H, —CH₂—),4.10 (s, 2H, —CH₂—), 3.79 (s, 3H, —OCH₃), 3.76 (s, 3H, —OCH₃), 3.30 (m,2H, —CH₂—), 2.92 (t, 2H, —CH₂—), 1.80 (s, 3H, —COCH₃). MS (FAB): 645(M+1).

Example 15N-(4-(2-bromo-3-(3,4-dimethoxyphenyl)benzyloxy)-2-(3-cyanobenzyloxy)benzyl)proline

The procedure was the same as in Example 11, except that proline wasused in place of 2-aminoethanol to afford an off-white solid powder. ¹HNMR (400 MHz, DMSO-d₆) δ 8.02 (s, 1H, —ArH), 7.90 (d, 1H, —ArH), 7.82(d, 1H, —ArH), 7.62 (t, 1H, —ArH), 7.56 (d, 1H, —ArH), 7.46 (t, 1H,—ArH), 7.37 (d, 2H, —ArH), 7.04 (d, 1H, —ArH), 6.96 (s, 1H, —ArH), 6.91(d, 1H, —ArH), 6.83 (s, 1H, —ArH), 6.69 (d, 1H, —ArH), 5.27 (m, 2H,—CH₂—), 5.19 (s, 2H, —CH₂—), 4.20-4.02 (m, 2H, —CH₂—), 3.81 (s, 3H,—OCH₃), 3.78 (s, 3H, —OCH₃), 3.55-3.49 (m, 1H, —CH—), 3.28-3.20 (m, 1H,—CH₂—), 2.85 (q, 1H, —CH₂—), 2.18-2.05 (m, 1H, —CH₂—), 2.02-1.94 (m, 1H,—CH₂—), 1.91-1.79 (m, 1H, —CH₂—), 1.77-1.61 (m, 1H, —CH₂—). MS (FAB):658 (M+1).

Example 16N-acetylaminoethyl-4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-2-(3-cyanobenzyloxy)benzylamine

(1) 2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl) toluene

The procedure was the same as in Example 1, except that2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolanewas used in place of phenylboronic acid, [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) was used in place oftriphenylphosphine palladium, potassium carbonate was used in place ofcesium carbonate to afford2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl) toluene as a pale yellowoil. ¹H NMR (400 MHz, Chloroform-d) δ 7.21 (d, 2H, —ArH), 7.11 (m, 1H,—ArH), 6.90 (d, 2H, —ArH), 6.86 (d, 1H, —ArH), 4.30 (m, 4H, —OCH₂CH₂O—),2.48 (s, 3H, —CH₃).

(2)4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-2-hydroxybenzaldehyde

The procedure was the same as in Example 1, except that2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl) toluene was used inplace of 2-Bromo-3-methyl-1,1′-biphenyl to effect bromination; thebromide, without further purification, was reacted directly with 2,4-dihydroxybenzaldehyde to afford a white solid. ¹H NMR (400 MHz,DMSO-d₆) δ 10.91 (s, 1H, —OH), 9.95 (s, 1H, —CHO), 7.57 (d, 1H, —ArH),7.45 (d, 1H, —ArH), 7.37 (t, 1H, —ArH), 7.25 (d, 1H, —ArH), 6.84 (d, 1H,—ArH), 6.78 (s, 1H, —ArH), 6.74 (d, 1H, —ArH), 6.59 (d, 1H, —ArH), 6.51(s, 1H, —ArH), 5.16 (s, 2H, —CH₂—), 4.20 (m, 4H, —OCH₂CH₂O—).

(3)4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-2-(3-cyanobenzyloxy) benzaldehyde

The procedure was the same as in Example 1, except that4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-2-hydroxybenzaldehyde was used in place of4-(2-bromo-3-phenylbenzyloxy)-2-hydroxybenzaldehyde to afford a whitesolid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.28 (s, 1H, —CHO), 8.01 (s, 1H,—ArH), 7.85 (dd, 2H, —ArH), 7.74 (d, 1H, —ArH), 7.63 (t, 1H, —ArH), 7.58(d, 1H, —ArH), 7.46 (t, 1H, —ArH), 7.35 (d, 1H, —ArH), 6.94 (d, 2H,—ArH), 6.87 (s, 1H, —ArH), 6.82 (d, 2H, —ArH), 5.36 (s, 2H, —CH₂—), 5.30(s, 2H, —CH₂—), 4.29 (m, 4H, —OCH₂CH₂O—).

(4)N-hydroxyethyl-4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-2-(3-cyanobenzyloxy)benzylamine

The procedure was the same as in Example 1, except that4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-2-(3-cyanobenzyloxy)benzaldehyde was used in place of4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzaldehyde to affordan off-white solid powder. ¹H NMR (400 MHz, DMSO-d₆) δ 8.74 (s, 1H,—NH—), 8.14 (m, 1H, —CONH—), 8.00 (s, 1H, —ArH), 7.85 (dd, 2H, —ArH),7.63 (t, 1H, —ArH), 7.54 (d, 1H, —ArH), 7.50-7.37 (m, 2H, —ArH), 7.33(d, 1H, —ArH), 6.94 (d, 1H, —ArH), 6.86 (s, 2H, —ArH), 6.82 (d, 1H,—ArH), 6.74 (d, 1H, —ArH), 5.27 (s, 2H, —CH₂—), 5.20 (s, 2H, —CH₂—),4.29 (m, 4H, —OCH₂CH₂O—), 4.13 (s, 2H, —CH₂—), 3.34-3.39 (m, 2H, —CH₂—),2.96 (m, 2H, —CH₂—), 1.82 (s, 3H, —COCH₃). MS (FAB): 643 (M+1).

Example 17N-(4-(2-bromo-3-(3,4-dimethoxyphenyl)benzyloxy)-2-(3-cyanobenzyloxy)benzyl)alanine

The procedure was the same as in Example 11, except that alanine wasused in place of 2-aminoethanol, to afford a white solid. ¹H NMR (400MHz, DMSO-d₆) δ 7.99 (s, 1H, —ArH), 7.89 (dd, 1H, —ArH), 7.80 (d, 1H,—ArH), 7.60 (t, 1H, —ArH), 7.53 (d, 1H, —ArH), 7.44 (t, 1H, —ArH), 7.35(d, 2H, —ArH), 7.02 (d, 1H, —ArH), 6.94 (s, 1H, —ArH), 6.89 (d, 1H,—ArH), 6.81 (d, 1H, —ArH), 6.67 (d, 1H, —ArH), 5.23 (s, 2H, —CH₂—), 5.17(s, 2H, —CH₂—), 3.94 (s, 1H, —CH₂—), 3.84 (s, 1H, —CH₂—), 3.79 (s, 3H,—OCH₃), 3.76 (s, 3H, —OCH₃), 3.35-3.38 (m, 1H, —CH—), 1.22 (s, 3H,—CH₃). MS (FAB): 632 (M+1).

Example 18N-(4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-2-(3-cyanobenzyloxy) benzyl) serine

The procedure was the same as in Example 16, except that serine was usedin place of 2-acetamidoethylamine, to afford an off-white solid powder.¹H NMR (400 MHz, DMSO-d₆) δ 8.01 (s, 1H, —ArH), 7.90 (d, 1H, —ArH), 7.82(d, 1H, —ArH), 7.61 (t, 1H, —ArH), 7.54 (d, 1H, —ArH), 7.44 (t, 1H,—ArH), 7.33 (t, 2H, —ArH), 6.93 (d, 1H, —ArH), 6.86 (s, 1H, —ArH), 6.82(s, 2H, —ArH), 6.68 (d, 1H, —ArH), 5.24 (s, 2H, —CH₂—), 5.18 (s, 2H,—CH₂—), 4.29 (s, 4H, —OCH₂CH₂O—), 4.04 (s, 2H, —NCH₂—), 3.73 (s, 1H,—NH—), 3.70-3.58 (m, 1H, —NCH—), 3.17 (s, 2H, —CH₂—). MS (FAB): 646(M+1).

Example 19N-(4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-2-(3-cyanobenzyloxy) benzyl) threonine

The procedure was the same as in Example 16, except that threonine wasused in place of 2-acetamidoethylamine to afford a white solid. ¹H NMR(400 MHz, DMSO-d₆) δ 7.96 (s, 1H, —ArH), 7.91-7.77 (m, 2H, —ArH), 7.62(t, 1H, —ArH), 7.54 (d, 1H, —ArH), 7.44 (t, 1H, —ArH), 7.32 (d, 1H,—ArH), 7.24 (s, 1H, —ArH), 6.93 (d, 1H, —ArH), 6.82 (m, 3H, —ArH), 6.64(d, 1H, —ArH), 5.22 (s, 2H, —CH₂—), 5.16 (s, 2H, —CH₂—), 4.29 (s, 4H,—OCH₂CH₂O—), 3.82 (s, 2H, —CH₂—), 3.35-3.38 (m, 1H, —CH—), 1.87 (s, 3H,—CH₃). MS (FAB): 660 (M+1).

Example 20:N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy) benzyl)serine

(1) 4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-hydroxybenzaldehyde

The procedure was the same as in Example 1, except that 2,4-dihydroxy-5-chloro-benzaldehyde was used in place of 2,4-dihydroxybenzaldehyde to afford a white solid. ¹H NMR (500 MHz,DMSO-d₆) δ 11.18 (s, 1H, —OH), 10.09 (s, 1H, —CHO), 7.74 (s, 1H, —ArH),7.66 (d, 1H, —ArH), 7.57 (t, 1H, —ArH), 7.51 (m, 2H, —ArH), 7.46 (d, 1H,—ArH), 7.42 (d, 3H, —ArH), 6.85 (s, 1H, —ArH), 5.37 (s, 2H, —CH₂—).

(2) 4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy)benzaldehyde

The procedure was the same as in Example 1, except that4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-hydroxybenzaldehyde was used inplace of 4-(2-bromo-3-phenylbenzyloxy)-2-hydroxybenzaldehyde to afford awhite solid. ¹H NMR (500 MHz, DMSO-d₆) δ 10.27 (s, 1H, —CHO), 8.07 (s,1H, —ArH), 7.91 (d, 1H, —ArH), 7.87 (d, 1H, —ArH), 7.77 (s, 1H, —ArH),7.73-7.64 (m, 2H, —ArH), 7.56 (m, 1H, —ArH), 7.51 (m, 2H, —ArH), 7.46(d, 1H, —ArH), 7.43 (m, 3H, —ArH), 7.25 (s, 1H, —ArH), 5.48 (s, 2H,—CH₂—), 5.46 (s, 2H, —CH₂—).

(3) N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy)benzyl) serine

The procedure was the same as in Example 1, except that4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy)benzaldehydewas used in place of4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzaldehyde, serinewas used in place of 2-acetamidoethylamine to afford an off-white solid.¹H NMR (500 MHz, DMSO-d₆) δ 8.02 (s, 1H, —ArH), 7.92 (d, 1H, —ArH), 7.84(d, 1H, —ArH), 7.65 (m, 2H, —ArH), 7.55 (d, 2H, —ArH), 7.52-7.47 (m, 2H,—ArH), 7.46 (d, 1H, —ArH), 7.41 (m, 3H, —ArH), 7.07 (s, 1H, —ArH), 5.33(s, 2H, —CH₂—), 5.31 (s, 2H, —CH₂—), 4.03 (s, 2H, —CH₂—), 3.83-3.63 (m,2H, —CH₂—), 3.38-3.43 (m, 1H, —CH—). MS (FAB): 622 (M+1).

Example 21N-hydroxylethyl-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy)benzylamine

The procedure was the same as in Example 1, except that4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy)benzaldehydewas used in place of4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzaldehyde,2-aminoethanol was used in place of 2-acetamidoethylamine to afford apale yellow solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.00 (s, 1H, —ArH), 7.86(d, 1H, —ArH), 7.82 (d, 1H, —ArH), 7.63 (q, 2H, —ArH), 7.56 (s, 1H,—ArH), 7.54-7.49 (m, 1H, —ArH), 7.47 (d, 2H, —ArH), 7.43 (d, 1H, —ArH),7.38 (d, 3H, —ArH), 7.07 (s, 1H, —ArH), 5.32 (s, 2H, —CH₂—), 5.30 (s,2H, —CH₂—), 3.97 (s, 2H, —CH₂—), 3.59 (t, 2H, —CH₂—), 2.81 (t, 2H,—CH₂—). MS (FAB): 679 (M+1).

Example 22N-acetylaminoethyl-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy)benzylamine

The procedure was the same as in Example 1, except that4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy)benzaldehydewas used in place of4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzaldehyde to affordan off-white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.00 (s, 1H, —ArH), 7.86(dd, 2H, —ArH), 7.69-7.62 (m, 2H, —ArH), 7.53 (d, 2H, —ArH), 7.50 (d,2H, —ArH), 7.46 (d, 1H, —ArH), 7.41 (t, 3H, —ArH), 7.07 (s, 1H, —ArH),5.33 (s, 2H, —CH₂—), 5.32 (s, 2H, —CH₂—), 3.89 (s, 2H, —CH₂—), 3.25 (m,2H, —CH₂—), 2.74 (t, 2H, —CH₂—), 1.83 (s, 3H, —COCH₃). MS (FAB): 620(M+1).

Example 23N-(2-methanesulfonylaminoethyl)-4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzylamine.Hydrochloride

N-(2-aminoethyl)methanesulfonamide-F₃CCOOH (76 mg) was dissolved in 5 mlDMF, and triethylamine (30 mg) was added. After stirring for 20 min,4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy) benzaldehyde (50 mg)and acetic acid (54 mg) were added. After stirring for 30 min, sodiumcyanoborohydride (15.7 mg) was added and the mixture was stirred at roomtemperature for 12 h. The reaction was stopped. The mixture wasextracted with water and ethyl acetate for three times. The organicphase was washed with saturated brine, and dried over anhydrous sodiumsulfate, then concentrated in vacuo. The crude residue was purified bysilica gel column chromatography to give a viscous product. 10 mL ofsaturational HCl methanol solution was added, and stirred overnight. Themixture was concentrated in vacuo, and washed with ether to afford apale yellow solid powder. ¹H NMR (400 MHz, DMSO-d₆) δ 9.06 (s, 2H,—NH—), 8.01 (s, 1H, —ArH), 7.89 (d, 1H, —ArH), 7.83 (d, 1H, —ArH),7.68-7.57 (m, 2H, —ArH), 7.50 (d, 1H, —ArH), 7.48 (d, 2H, —ArH), 7.45(s, 1H, —ArH), 7.42 (s, 1H, —ArH), 7.39 (m, 3H, —ArH), 6.86 (d, 1H,—ArH), 6.75 (d, 1H, —ArH), 5.28 (s, 2H, —CH₂—), 5.22 (s, 2H, —CH₂—),4.14 (t, 2H, —CH₂—), 3.30 (m, 2H, —CH₂—), 3.02 (m, 2H, —CH₂—), 2.94 (s,3H, —CH₃). MS (FAB): 658 (M+1).

Example 24(S)—N-(4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-5-chloro-2-(3-cyanobenzyloxy)benzyl)pipecolinic acid

The procedure was the same as in Example 16, except that 2,4-dihydroxy-5-chloro-benzaldehyde was used in place of 2,4-dihydroxybenzaldehyde, and pipecolinic acid was used in place of2-acetamidoethylamine to afford an off-white solid powder. ¹H NMR (400MHz, DMSO-d₆) δ 7.94 (s, 1H, ArH), 7.81 (m, 2H, ArH), 7.61 (t, J=8.0 Hz,2H, ArH), 7.46 (m, 2H, ArH), 7.34 (d, J=7.6 Hz, 1H, ArH), 7.01 (s, 1H,ArH), 6.94 (d, J=8.4 Hz, 1H, ArH), 6.89-6.79 (m, 2H, ArH), 5.28 (s, 2H,—CH₂—), 5.25 (s, 2H, —CH₂—), 4.29 (s, 4H, —CH₂—), 3.70 (dd, J₁=13.6 Hz,J₂=52.4 Hz, 2H, —CH₂—), 3.22-3.12 (m, 1H, —CH—), 2.91 (m, 1H, —CH₂—),2.31 (m, 1H, —CH₂—), 1.76 (m, 2H, —CH₂—), 1.49 (br s, 3H, —CH₂—), 1.38(br s, 1H, —CH₂—). MS (FAB): 705 (M+1).

Example 25N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy)benzyl)alanine

The procedure was the same as in Example 1, except that4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy)benzaldehydewas used in place of4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzaldehyde, andalanine was used in place of 2-acetamidoethylamine to affordN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy)benzyl)alanine as a white solid. MS (FAB): 606 (M).

Example 26:N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy) benzyl)threonine

The procedure was the same as in Example 1, except that4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy)benzaldehydewas used in place of4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzaldehyde, andthreonine was used in place of 2-acetamidoethylamine to affordN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy)benzyl)threonine as a white solid. MS (FAB): 636 (M).

Example 27N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-methanesulfonylbenzyloxy)benzyl) serine

The procedure was the same as in Example 1, except that4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-hydroxybenzaldehyde was used inplace of 4-(2-bromo-3-phenylbenzyloxy)-2-hydroxybenz aldehyde,3-methanesulfonylbenzyl bromide was used in place of 3-cyanobenzylbromide, and serine was used in place of 2-acetamidoethylamine to affordN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-methanesulfonylbenzyloxy) benzyl) serine as a solidpowder. MS (FAB): 675 (M).

Example 28N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-methanesulfonylbenzyloxy)benzyl) pipecolinic acid

The procedure was the same as in Example 1, except that4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-hydroxybenzaldehyde was used inplace of 4-(2-bromo-3-phenylbenzyloxy)-2-hydroxy benzaldehyde,3-methanesulfonylbenzyl bromide was used in place of 3-cyanobenzylbromide, and pipecolinic acid was used in place of 2-acetamidoethylamineto affordN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-methanesulfonylbenzyloxy)benzyl)pipecolinic acid as a solid powder. MS (FAB): 699 (M).

Pharmacological Experiments

1. In vitro activity evaluation: Cisbio PD-1/PD-L1 binding assay kit wasapplied for the detection method of in vitro enzymology level.

Screening Principles and Methods of PD-1/PD-L1 Small Molecule Inhibitors

1) Principle: PD-1 protein is with HIS tag, and PD-1 ligand PD-L1 iswith hFc tag. Eu labeled anti-hFc antibody and XL665 labeled anti-HISantibody are combined with the above two label proteins respectively.After laser excitation, energy can be transferred from donor Eu toreceptor XL665, allowing XL665 to glow. After adding inhibitors(compounds or antibodies), blocking the binding of PD-1 and PD-L1 makesthe distance between Eu and XL665 far away, the energy can not betransferred, and XL665 does not glow.

2) Experimental method: The specific method can be referred to Cisbio'sPD-1/PD-L1 Kit (item 64CUS000C-2). Reagents should be dispensed in thefollowing order. For 384-well white ELISA plate, 2 μl of diluent ortarget compound diluted with diluent was added to each well, and then 4μl of PD-1 protein and 4 μl of PD-L1 protein were added per well,incubated for 15 min at room temperature; and 10 μl of a mixture ofanti-Tag1-Eu3⁺ and anti-Tag2-XL665 was added per well and incubated for1 h to 4 h at room temperature and the fluorescence signals at 665 nmand 620 nm were measured with an Envison instrument. HTRF rate=(665nm/620 nm)*10⁴. 8-10 concentrations were detected for each compound andIC₅₀ was calculated by Graphpad software.

3) The results of the screening were shown in Table 1.

TABLE 1 Evaluation of the inhibitory activity of the example compoundsat molecular level on the interaction between PD-1 and PD-L1: ExampleIC₅₀ (M) Example IC₅₀ (M) 1 4.39 × 10⁻⁷ 15 — 2 2.94 × 10⁻⁸ 16 2.68 ×10⁻⁷ 3 2.30 × 10⁻⁷ 17 3.16 × 10⁻⁶ 4 5.92 × 10⁻⁸ 18 3.85 × 10⁻⁸ 5 3.07 ×10⁻⁷ 19 1.81 × 10⁻⁸ 6 1.78 × 10⁻⁷ 20 2.48 × 10⁻⁹ 7 4.59 × 10⁻⁶ 21 5.29 ×10⁻⁹ 8 1.34 × 10⁻⁷ 22 6.23 × 10⁻⁸ 9 7.88 × 10⁻⁷ 23 9.79 × 10⁻⁷ 10 4.58 ×10⁻⁷ 24 6.75 × 10⁻⁸ 11 7.87 × 10⁻⁶ 25 3.16 × 10⁻⁹ 12 — 26 ≤10⁻⁸ 13 5.35× 10⁻⁶ 27 ≤10⁻⁸ 14 6.65 × 10⁻⁶ 28 ≤10⁻⁸ Cisbio HTRF detection showedthat the interaction of PD-1 and PD-L1 could be significantly inhibitedby the example compounds at the molecular level, with IC₅₀ < 10⁻¹³mol/L.

2. The Example Compounds' Capacity of Relieving the Inhibition of IFNγby Ligand PD-L1:

The expression level of IFNγ can reflect the proliferative activity of Tlymphocytes. Using the extracted human PBMC (peripheral bloodmononuclear cell), on the basis that T lymphocyte could be activated bythe anti-CD3/anti-CD28 antibody, the ligand PD-L1 was added to theinhibit T lymphocyte, the example compounds' capacity of relieving theinhibition by the PD-L1 was investigated.

The specific procedure is as follows. DAKEWE human lymphocyte separationsolution (DKW-KLSH-0100) was used to extract PBMC from human wholeblood, and PBMC was inoculated into 96 well plate, with 3×10⁵ cells perwell. Human PD-L1 protein (final concentration 5 μg/ml),anti-CD3/anti-CD28 antibody (final concentration 1 μg/ml) andproportional dilution of the example compounds were added respectively.After 72 h, the expression level of IFNγ in the supernatant was detectedby Cisbio IFNγ test kit. The experimental results showed that theinhibition of PD-L1 to expression level of IFNγ could be partiallyrelieved by the example compounds at 10 nM.

3. The Efficacy of the Example Compounds In Vivo

The methods of pharmacodynamics were as follows:

The method in subcutaneous xenograft tumor was as follows. The culturedspecific tumor cells were digested and collected by centrifugation, andwashed with sterile physiological saline for two times and then counted.The cell concentration was adjusted to 5×10⁶/ml by physiological saline,and 0.2 ml of cell suspension was inoculated to the right armpit ofC57BL/6 or Bablc mice. After inoculation, the animals were randomlydivided into two groups in next day. Each group had 6-7 mice. Afterweighing, the animals were dosed once each day to monitor tumor size.When the tumor size reached to a certain size, the mice was weighed andblood was collected from mice orbit and then the mice were killed byremoving the neck. The tumor tissue, thymus tissue and spleen tissuewere collected and weighed respectively. Finally, the tumor growthinhibition rate was calculated, and the tumor growth inhibition rate wasused to evaluate the level of anti-tumor effect.

The method in B16F10 lung metastasis model was as follows. The culturedB16F10 tumor cells were digested and centrifuged and washed for twotimes with sterile physiological saline and then counted. And the cellconcentration was adjusted to 2.5×10⁶/ml by physiological saline. 0.2 mlof cells were injected into the C57BL/6 mice through the tail vein, andthe tumor cells will gather in the lung of the mice. After inoculation,the animals were randomly divided into two groups in next day. Eachgroup had 6-7 mice. After weighing, the animals were dosed once eachday. After 3 weeks, the mice were weighed and killed, the lung tissuewas collected and weighed, and the number of lung tumors was countedafter being fixed by the Bouin's Fluid. Finally, the tumor growthinhibition rate was calculated, and the tumor growth inhibition rate wasused to evaluate the level of anti-tumor effect.

The method in Lewis lung cancer hydrothorax model was as follows: Thesubcutaneous xenograft tumor of Lewis lung cancer was homogenized andwashed for two times with sterile physiological saline, and the cellconcentration was adjusted to 2.5×10⁵/ml by physiological saline. 0.2 mlof cells were injected into the thoracic cavity of C57BL/6 mice. Afterinoculation, the animals were randomly divided into two groups in nextday. Each group had 6-7 mice. After weighing, the animals were dosedonce each day. Animals were sacrificed when the weight of the animals inthe control group suddenly dropped. The liquid in thoracic cavity wasextracted with syringe and the volume of fluid was recorded.

In the study of the mechanism of the above models, the method of flowcytometry was adopted in measuring the total cell proportion of T cellsof various types. The specific steps were as follows. The samples weretreated at first. For blood tissue, the orbital blood was taken. The redcell lysate was used to remove the red blood cells, and then the PBSbuffer was used for wash. After being washed, the cells were collected.For the tumor and spleen, the tissues were grinded with a homogenizer,and then diluted with PBS buffer, then filtered by 300 meshes of screen.After the number of cells was counted for each sample, 1×10⁶ cells wereadded into EP tube and stained for flow antibody. After incubation for 1h on ice, each sample was washed 2 times with PBS buffer. The cellpopulation was analyzed by VERSE flow instrument of BD Company. Thetotal number of cells in tumor tissue was 1×10⁵ and the total number ofcells in blood and spleen tissues was 1×10⁴. The ratio of T cells tototal number of cells was analyzed after flow cytometry.

(1) Subcutaneous Xenograft Model of High Metastatic Melanoma B16F10

For the high metastatic melanoma B16F10, the example compounds cansignificantly inhibit the growth of the subcutaneous tumor, with therespect of tumor volume or weight.

From the analysis of mechanism, the example compounds can increase theproportion of tumor-infiltrating lymphocytes and the proportion oflymphocytes in the spleen.

(2) Lung metastasis model of high metastatic melanoma B16F10 Formetastatic lung cancer models with high metastatic melanoma B16F10, theexample compounds can significantly inhibit the number of lungmetastases. From analysis of the mechanism, the example compounds canincrease the percentage of lymphocyte in mouse blood.

(3) Subcutaneous Xenograft Model of Mouse Breast Cancer EMT6

For subcutaneous xenograft model of mouse breast cancer EMT6, theexample compounds have some inhibition effect on mouse breast cancerEMT6, and the combination of the example compounds and CTX cansignificantly increase the tumor growth inhibition rate of CTX.

(4) Mouse Lewis Lung Cancer Hydrothorax Model

The example compounds have significant inhibition effect on mouse Lewislung hydrothorax model, and can reduce the hydrothorax incidence rate.

(5) Subcutaneous Xenograft Model of Mouse Colon Cancer MC38

For subcutaneous xenograft model of mouse colon cancer MC38, the examplecompounds have significant inhibition effect on mouse colon cancer MC38,and have a synergistic antitumor effect on this cancer in combinationwith CTX.

4. The Interaction of Example Compound/PD-L1 Antibody with PD-L1 Proteinwas Tested by Biacore

(1) Experimental principle

Surface plasmon is a kind of electromagnetic wave on the surface ofmetal, produced by the interaction of photon and electron in freevibration. Surface plasmon resonance (SPR) is an optical phenomenon thatoccurs on the surface of two kinds of media, which can be induced byphoton or electron. The phenomenon of total reflection of light fromlight dense medium into light scattering medium will form evanescentwave into light scattering medium. When the total reflected evanescentwave meets the plasma wave on the metal surface, the resonance mayoccur, and the energy of reflected light decreases and the resonancepeak appears on the reflected light energy spectrum. This resonance iscalled the surface plasmon resonance. The incident angle of the surfaceplasmon resonance is called the SPR angle. The SPR biosensor provides asensitive, real-time, non-label detection technique for monitoring theinteraction of molecules. The sensor detects the change of the SPRangle, and SPR is also related to the refractive index of the metalsurface. When an analyte is bond on the surface of the chip, it leads tothe change of the refractive index of the chip surface, which leads tothe change of the SPR angle. This is the basic principle of thereal-time detection of intermolecular interaction by the SPR biosensor.In the interaction analysis, the change of SPR angle is recorded on thesensor map in real time.

(2) Experimental Methods

The PD-L1 protein was captured on the Fc4 channel of NTA chip by capturemethod, and the buffer system was PBS-P+, pH7.4, 0.01% DMSO. A series ofconcentration of compounds and PD-L1 antibodies were prepared and flowedthrough the surface of the chip for the determination of interaction.

(3) Experimental Results

It was preliminarily determined that the binding protein of the examplecompounds was PD-L1. Further Biacore experiments confirmed that theexample compounds had a strong ability of binding PD-L1.

What is claimed is:
 1. A benzyl phenyl ether derivative of Formula (I):

or a stereoisomer or a pharmaceutically acceptable salt thereof,wherein: R₁ is selected from

R₂ is selected from hydrogen, cyano, methylsulfonyl, acetylamino,carbamoyl, dimethyl amino formyl (—CON(CH₃)₂), fluorine, chlorine,bromine, iodine, trifluoromethyl, C₁-C₅ alkyh and C₁-C₅ alkoxy; R₃ isselected from substituted C₁-C₈ saturated alkylamino, substituted C₂-C₆unsaturated alkylamino, and substituted N-containing C₂-C₆heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substitutedwith substituent(s) selected from hydrogen, fluorine, chlorine, bromine,iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino, C₁-C₆ alkylamino,acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino (—NH(C═NH)NH₂),ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂),sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino(—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxyl carbonyl, sulfydryl,imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.
 2. A benzyl phenyl etherderivative of claim 1, represented by formula (IA), or apharmaceutically acceptable salt or a stereoisomer thereof:

wherein: R₁ is selected from

R₂ is selected from hydrogen, cyano, methylsulfonyl, acetylamino,carbamoyl, dimethyl amino formyl (—CON(CH₃)₂), fluorine, chlorine,bromine, iodine, trifluoromethyl, C₁-C₅ alkyl, and C₁-C₅ alkoxy; R₃ isselected from substituted C₁-C₈ saturated alkylamino, substituted C₂-C₆unsaturated alkylamino, and substituted N-containing C₂-C₆heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substitutedwith substituent(s) selected from hydrogen, fluorine, chlorine, bromine,iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino, C₁-C₆ alkylamino,acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino (—NH(C═NH)NH₂),ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂),sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino(—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxyl carbonyl, sulfydryl,imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.
 3. A benzyl phenyl etherderivative of claim 2, represented by formula (IA-1), or apharmaceutically acceptable salt or a stereoisomer thereof:

wherein: R₂ is selected from hydrogen, cyano, methylsulfonyl,acetylamino, carbamoyl, dimethyl amino formyl (—CON(CH₃)₂), fluorine,chlorine, bromine, iodine, trifluoromethyl, C₁-C₅ alkyl and C₁-C₅alkoxy; R₃ is selected from substituted C₁-C₈ saturated alkylamino,substituted C₂-C₆ unsaturated alkylamino, and substituted N-containingC₂-C₆ heterocycle-1-yl, wherein each is mono-, di-, tri-, ortetra-substituted with substituent(s) selected from hydrogen, fluorine,chlorine, bromine, iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino,C₁-C₆ alkylamino, acetylamino, cyano, ureido (—NH(C═O)NH₂), g(—NH(C═NH)NH₂), ureido amino (—NH—NH(C═O)NH₂), guanidino amino(—NH—NH(C═NH)NH₂), sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂),methanesulfonylamino (—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxylcarbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.
 4. A benzyl phenyl etherderivative of claim 3, represented by formula (IA-1a), or apharmaceutically acceptable salt or a stereoisomer thereof:

wherein: R₃ is selected from substituted C₁-C₈ saturated alkylamino,substituted C₂-C₆ unsaturated alkylamino, and substituted N-containingC₂-C₆ heterocycle-1-yl, wherein each is mono-, di-, tri-, ortetra-substituted with substituent(s) selected from hydrogen, fluorine,chlorine, bromine, iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino,C₁-C₆ alkylamino, acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino(—NH(C═NH)NH₂), ureido amino (—NH—NH(C═O)NH₂), guanidino amino(—NH—NH(C═NH)NH₂), sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂),methanesulfonylamino (—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxylcarbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.
 5. A benzyl phenyl etherderivative of claim 3, represented by formula (IA-1b), or apharmaceutically acceptable salt or a stereoisomer thereof:

wherein: R₃ is selected from substituted C₁-C₈ saturated alkylamino,substituted C₂-C₆ unsaturated alkylamino, and substituted N-containingC₂-C₆ heterocycle-1-yl, wherein each is mono-, di-, tri-, ortetra-substituted with substituent(s) selected from hydrogen, fluorine,chlorine, bromine, iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino,C₁-C₆ alkylamino, acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino(—NH(C═NH)NH₂), ureido amino (—NH—NH(C═O)NH₂), guanidino amino(—NH—NH(C═NH)NH₂), sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂),methanesulfonylamino (—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxylcarbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.
 6. A benzyl phenyl etherderivative of claim 2, represented by formula (IA-2), or apharmaceutically acceptable salt or a stereoisomer thereof:

wherein: R₂ is selected from hydrogen, cyano, methylsulfonyl,acetylamino, carbamoyl, dimethyl amino formyl (—CON(CH₃)₂), fluorine,chlorine, bromine, iodine, trifluoromethyl, C₁-C₅ alkyl and C₁-C₅alkoxy; R₃ is selected from substituted C₁-C₈ saturated alkylamino,substituted C₂-C₆ unsaturated alkylamino, and substituted N-containingC₂-C₆ heterocycle-1-yl, wherein each is mono-, di-, tri-, ortetra-substituted with substituent(s) selected from hydrogen, fluorine,chlorine, bromine, iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino,C₁-C₆ alkylamino, acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino(—NH(C═NH)NH₂), ureido amino (—NH—NH(C═O)NH₂), guanidino amino(—NH—NH(C═NH)NH₂), sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂),methanesulfonylamino (—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxylcarbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.
 7. A benzyl phenyl etherderivative of claim 6, represented by formula (IA-2a), or apharmaceutically acceptable salt or a stereoisomer thereof:

wherein: R₃ is selected from substituted C₁-C₈ saturated alkylamino,substituted C₂-C₆ unsaturated alkylamino, and substituted N-containingC₂-C₆ heterocycle-1-yl, wherein each is mono-, di-, tri-, ortetra-substituted with substituent(s) selected from hydrogen, fluorine,chlorine, bromine, iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino,C₁-C₆ alkylamino, acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino(—NH(C═NH)NH₂), ureido amino (—NH—NH(C═O)NH₂), guanidino amino(—NH—NH(C═NH)NH₂), sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂),methanesulfonylamino (—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxylcarbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.
 8. A benzyl phenyl etherderivative of claim 6, represented by formula (IA-2b), or apharmaceutically acceptable salt or a stereoisomer thereof:

wherein: R₃ is selected from substituted C₁-C₈ saturated alkylamino,substituted C₂-C₆ unsaturated alkylamino, and substituted N-containingC₂-C₆ heterocycle-1-yl, wherein each is mono-, di-, tri-, ortetra-substituted with substituent(s) selected from hydrogen, fluorine,chlorine, bromine, iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino,C₁-C₆ alkylamino, acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino(—NH(C═NH)NH₂), ureido amino (—NH—NH(C═O)NH₂), guanidino amino(—NH—NH(C═NH)NH₂), sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂),methanesulfonylamino (—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxylcarbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.
 9. A benzyl phenyl etherderivative of claim 1, or a pharmaceutically acceptable salt or astereoisomer thereof, wherein R₃ is of one of the following formulae:

wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl,pentyl, hexyl, heptyl, and octyl; and X is selected from hydrogen,fluorine, chlorine, bromine, methyl, ethenyl, and trifluoromethyl.
 10. Abenzyl phenyl ether derivative of claim 1, or a pharmaceuticallyacceptable salt or a stereoisomer thereof, wherein the compound isselected from:N-acetylaminoethyl-4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzylamine

N-(4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy) benzyl) serine

N-Ethyl-N-hydroxylethyl-4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzylamine

N-(4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy) benzyl) proline

N-hydroxylethyl-4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzylamine

N-(4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy) benzyl) alanine

N-(4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy) benzyl) methionine

N-(4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy) benzyl) threonine

N-(tetrahydro-2H-pyran-4-yl)-4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzylamine

N-[4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy) benzyl] morpholineHydrochloride

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy) benzyl)serine

N-hydroxylethyl-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy)benzylamine

N-acetylaminoethyl-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy)benzylamine

N-(2-methanesulfonylaminoethyl)-4-(2-bromo-3-phenylbenzyloxy)-2-(3-cyanobenzyloxy)benzylamine Hydrochloride

(S)-N-(4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-5-chloro-2-(3-cyanobenzyloxy)benzyl) pipecolinic acid

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy) benzyl)alanine

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-cyanobenzyloxy) benzyl)threonine

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-methanesulfonylbenzyloxy)benzyl) serine

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-methanesulfonylbenzyloxy)benzyl)pipecolinicacid


11. A benzyl phenyl ether derivative of claim 1, or a stereoisomer or apharmaceutically acceptable salt thereof, wherein the pharmaceuticallyacceptable salt comprises a salt formed with an inorganic acid, a saltformed with an organic acid, alkali metal ion salt, alkaline earth metalion salt, or a salt formed with organic base which provides aphysiologically acceptable cation, and an ammonium salt.
 12. A benzylphenyl ether derivative of claim 11, or a stereoisomer or apharmaceutically acceptable salt thereof, wherein the inorganic acid isselected from hydrochloric acid, hydrobromic acid, phosphoric acid, andsulfuric acid; the organic acid is selected from methanesulfonic acid,p-toluenesulfonic acid, trifluoroacetic, citric acid, maleic acid,tartaric acid, fumaric acid, citric acid, and lactic acid; the alkalimetal ion is selected from lithium ion, sodium ion, and potassium ion;the alkaline earth metal ion is selected from calcium ion and magnesiumion; and the organic base which provides a physiologically acceptablecation is selected form methylamine, dimethylamine, trimethylamine,piperidine, morpholine, and tris(2-hydroxyethyl) amine.
 13. A benzylphenyl ether derivative of claim 2, or a pharmaceutically acceptablesalt or a stereoisomer thereof, wherein R₃ is of one of the followingformulae:

wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl,pentyl, hexyl, heptyl, and octyl; and X is selected from hydrogen,fluorine, chlorine, bromine, methyl, ethenyl, and trifluoromethyl.
 14. Abenzyl phenyl ether derivative of claim 3, or a pharmaceuticallyacceptable salt or a stereoisomer thereof, wherein R₃ is of one of thefollowing formulae:

wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl,pentyl, hexyl, heptyl, and octyl; and X is selected from hydrogen,fluorine, chlorine, bromine, methyl, ethenyl, and trifluoromethyl.
 15. Abenzyl phenyl ether derivative of claim 4, or a pharmaceuticallyacceptable salt or a stereoisomer thereof, wherein R₃ is of one of thefollowing formulae:

wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl,pentyl, hexyl, heptyl, and octyl; and X is selected from hydrogen,fluorine, chlorine, bromine, methyl, ethenyl, and trifluoromethyl.
 16. Abenzyl phenyl ether derivative of claim 5, or a pharmaceuticallyacceptable salt or a stereoisomer thereof, wherein R₃ is of one of thefollowing formulae:

wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl,pentyl, hexyl, heptyl, and octyl; and X is selected from hydrogen,fluorine, chlorine, bromine, methyl, ethenyl, and trifluoromethyl.
 17. Abenzyl phenyl ether derivative of claim 6, or a pharmaceuticallyacceptable salt or a stereoisomer thereof, wherein R₃ is of one of thefollowing formulae:

wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl,pentyl, hexyl, heptyl, and octyl; and X is selected from hydrogen,fluorine, chlorine, bromine, methyl, ethenyl, and trifluoromethyl.
 18. Abenzyl phenyl ether derivative of claim 7, or a pharmaceuticallyacceptable salt or a stereoisomer thereof, wherein R₃ is of one of thefollowing formulae:

wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl,pentyl, hexyl, heptyl, and octyl; and X is selected from hydrogen,fluorine, chlorine, bromine, methyl, ethenyl, and trifluoromethyl.
 19. Abenzyl phenyl ether derivative of claim 8, or a pharmaceuticallyacceptable salt or a stereoisomer thereof, wherein R₃ is of one of thefollowing formulae:

wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl,pentyl, hexyl, heptyl, and octyl; and X is selected from hydrogen,fluorine, chlorine, bromine, methyl, ethenyl, and trifluoromethyl.
 20. Apharmaceutical composition, characterized in that it comprises a benzylphenyl ether derivative of claim 1, or a stereoisomer or apharmaceutically acceptable salt thereof, as an active ingredient, andone or more pharmaceutically acceptable carriers or excipients.
 21. Apharmaceutical composition, characterized in that it comprises a benzylphenyl ether derivative of claim 10, or a stereoisomer or apharmaceutically acceptable salt thereof, as an active ingredient, andone or more pharmaceutically acceptable carriers or excipients.
 22. Aprocess for the preparation of a benzyl phenyl ether derivative of claim1, or a stereoisomer or a pharmaceutically acceptable salt thereof,comprising the following steps:

(a) 2-hydroxy-4-(2-bromo-3-R1 benzyloxy)-X-substituted benzaldehyde 1 asa starting material is reacted with a benzyl halide which isR2-substituted at 3-position under basic conditions to obtain analdehyde-containing intermediate compound 2; (b) the aldehyde-containingintermediate compound 2 as the starting material is condensed with anamino group- or an imino group-containing HR3 and the resultant productis reduced to obtain a target compound I; wherein R₁, R₂, R₃ and X eachis defined as claim
 1. 23. A method for treating a disease associatedwith the PD-1/PD-L1 signaling pathway in a subject in need of suchtreatment comprising administering to the subject an effective amount ofa benzyl phenyl ether derivative of claim 1, or a stereoisomer thereof,or a pharmaceutically acceptable salt thereof; wherein the disease isselected from the group consisting of melanoma, lung cancer, breastcancer, and colon cancer.
 24. A method for treating a disease associatedwith the PD-1/PD-L1 signaling pathway in a subject in need of suchtreatment comprising administering to the subject an effective amount ofthe benzyl phenyl ether derivative of claim 10, or a stereoisomerthereof, or a pharmaceutically acceptable salt thereof; wherein thedisease is selected from the group consisting of melanoma, lung cancer,breast cancer, and colon cancer.